tRNA 2024

Transfer RNA (tRNA) modifications are essential for ensuring accurate and efficient translation. The physiological importance of tRNA modification has been demonstrated by human diseases caused by aberrant tRNA modification. Steady-state level of each tRNA and its modification status are dynamically regulated in different cells and tissues in spatiotemporal manner. Alteration of tRNA abundance and its modification frequency modulate optimal translation, affecting mRNA stability and/or proper protein folding. Nanopore sequencing has emerged as a powerful technology for directly analyzing RNA molecules by measuring ion current signals as the RNA strand passes through a nanopore protein. This technique can detect modified nucleotides by comparing their distinct current signals to those of unmodified ones. Typically, some RNA modifications can be detected by a basecaller. However, applying this method to tRNA modifications is challenging due to their complex chemical nature and dense clustering within short RNA segments. We have developed a novel computational approach named “signal alignment” that enables direct comparison of current signals without the need for mapping to a reference sequence. We first generate a reference signal from the current signals of both modified and unmodified RNAs, and then align all current signals to this reference. Modified nucleotides are detected based on the unique features of the aligned signals defined by current value, duration, and standard deviation at each aligned position. tRNA modifications are successfully clustered by these features and accurately quantified by a Gaussian mixture model. We applied this method to E. coli, yeast and human tRNAs and detected most of tRNA modifications. This approach enables us to classify and quantify a couple of complex tRNA modifications bearing multiple intermediates.

Use of Nanopore direct RNA sequencing for full-length tRNA sequencing is well documented (Thomas et al. 2021;Lucas et al. 2023;Suzuki, London Calling 2023;White et al. 2024;Shaw et al. 2024). Recently, Oxford Nanopore (ONT) released an updated Nanopore direct RNA sequencing platform (RNA004) with a substantially higher accuracy (98.3% for poly(A) RNA in our hands), and higher throughput. We applied RNA004 sequencing to human mitochondrial RNA from lymphoblastoid and embryonic stem cell lines.

tRNA reads were acquired as either part of three RNA classes (tRNA + rRNA + mRNA) or using exclusively tRNA-specific adapters. In the latter case, we acquired 13 million+ tRNA reads, including 1 million+ mitochondrial tRNA reads, using PromethION flow cells. The median basecall identity was ~92%. The accuracy is lower than for poly(A) RNA due to the abundant modifications in tRNA. By comparison, median basecall identity for synthetic canonical mt-tRNA (IVT-derived) was higher for over 1 million+ aligned reads. Using these data, we were able to document alignments to all 22 known human mitochondrial tRNA isoacceptors. Partially processed, polycistronic mitochondrial RNA strands were also observed. We will discuss how these results pertain to systematic sorting of mt-tRNA isotypes, principled base modification analysis, and their utility for detection of aberrant tRNA in disease.

Oxford Nanopore’s unique direct RNA sequencing chemistry enables sequencing RNA transcripts without converting to DNA. It is also possible to sequence modified RNA bases; information that would otherwise be lost with cDNA methods. We present improvements to our direct RNA sequencing chemistry (SQK-RNA004), with higher accuracy and output, and a software upgrade for obtaining mappable reads as short as ≥ 50 nt enabling sequencing of shorter RNA including tRNA. Lastly, we demonstrate modified base detection improvements, including models for m6A and psuedouridine (Ψ) at >97% accuracy, and the detection of various other modified bases, particularly those present in tRNA molecules.